Recent genetic studies in mice and humans have indicated that signaling through calcineurin and NFAT might be modulated to affect a wide variety of disease processes including diabetes, heart disease, osteoporosis, schizophrenia, immune deficiency and autoimmunity. Moreover we have recently shown that many biologic processes are exquisitely sensitive to the level of nuclear occupancy of NFATc proteins. These findings raise the possibility that small molecule modulators of NFATc nuclear occupancy might be valuable therapeutics. NFAT signaling is initiated when Ca2+ entry activates the phosphatase calcineurin, which in turn dephosphorylates NFATc proteins leading to their nuclear entry. In the nucleus NFATc proteins assemble on DNA with nuclear partner proteins (NFATn's) and form active transcription complexes. NFATc proteins are actively exported from the nucleus after sequential phosphorylation by the kinases Dyrk1a and GSK3. The process of rapid nuclear import and export is regulated by an uncharacterized allosteric switch in the N-terminus of the protein, which regulates the alternate interaction between NFATc proteins and the nuclear import and export machinery. Minor modifications of this switch can change nuclear occupancy and induce dramatic phenotypic changes. In the past allosteric switches have been effective drug and small molecule targets. Hence, this switch represents an ideal site for intervention by small molecules. We will develop a new assay for small molecule modulators of the nuclear import/export switch. We propose to develop high throughput screening (HTS) assays that target the conformational change and the key protein-protein interaction required for cytoplasmic-to-nuclear translocation of NFATc proteins. The protein-protein interactions, which we will assay are the interaction between the NFATc family of transcription factors and the karyopherin ?2 (importin?, KPNA2) and exportin (Crm1, XPO1). [unreadable] The objective of the first specific aim proposed in this application is to develop an in vitro multianalyte time resolved fluorescence (TRF) assay, which allows a quantitative assessment of the interaction of recombinant NFATc protein with the nuclear import and export machinery. The goal of the second specific aim of our proposal is to optimize and automate this assay for high-throughput screening (HTS) and use them in the pilot screen to discover small molecule modulators of calcineurin/NFAT signaling. The TRF assay will be complemented by two cell-based secondary assays, where the target is screened in a more physiological context than in the biochemical primary assays. The secondary assays will be used to establish the specificity and efficacy of small molecule modulators of NFATc cytoplasmic-to-nuclear translocation and NFAT dependent transcription. We are aiming at optimizing these secondary assays also for a HTS environment. We will use the 1,280 compounds in the library of pharmacologically active compounds (LOPAC) in a quantitative high throughput format to identify technical problems and confirm the feasibility of a larger scale high throughput screen. If the aims of this application are achieved, future implementation of the HTS assays in collaboration the MLSCN should identify novel compounds, which modulate NFAT transcriptional activity. Active compounds will [unreadable] be valuable chemical tools to study this critical signaling pathway and have the potential to be active in a variety of disease processes. Given the need for new therapeutic approaches in diseases where [unreadable] calcineurin /NFAT signaling is an important target, active compounds identified by the proposed assay would have a high probability of stimulating additional pharmaceutical development. [unreadable] [unreadable] [unreadable]